Systematic induced resistance in Solanum lycopersicum (L.) against vascular wilt pathogen (Fusarium oxysporum f. sp. lycopersici) by Citrullus colocynthis and Trichoderma viride

The antifungal effects of Citrullus colocynthis extract (Hexane, chloroform, methanol, and water) were tested in vitro on Fusarium oxysporum f. sp. lycopersici (Sacc.) W. C. Snyder & H. N. Hans (FOL), the causal agent of Fusarium wilt. Of these, methanol and water extract at 10% showed the highest inhibition of mycelial growth of FOL by 12.32 and 23.61 mm respectively. The antifungal compounds were identified through Fourier transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectroscopy (GC-MS). The methanol extract was compatible with the biocontrol agent Trichoderma viride. The antagonistic fungi were mass-cultured under laboratory conditions using sorghum seeds. Both T. viride and C. colocynthis methanol extract was also tested alone and together against FOL under both in vitro and in vivo conditions. The combination of T. viride and C. colocynthis showed the highest percentage of antifungal activity (82.92%) against FOL under in vitro conditions. This study revealed that induced systemic resistance (ISR) in enhancing the disease resistance in tomato plants against Fusarium wilt disease. The combined treatment of T. viride and C. colocynthis significantly reduced the disease incidence and index by 21.92 and 27.02% in greenhouse conditions, respectively. Further, the induction of defense enzymes, such as peroxidase (PO), polyphenol oxidase (PPO), β-1,3-glucanase, and chitinase were studied. The accumulation of defense enzyme was greater in plants treated with a combination of T. viride and C. colocynthis compared to the control. Reduction of wilt disease in tomato plants due to the involvement of defense-related enzymes is presumed through this experiment.

a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 combination of T. viride and C. colocynthis compared to the control. Reduction of wilt disease in tomato plants due to the involvement of defense-related enzymes is presumed through this experiment.
Trichoderma species are a beneficial cosmopolitan group of fungi that are predominant in soils across different ecosystems [22]. In addition to colonizing roots, these fungi attack, parasitize and suppress other pathogenic fungi [23,24]. Trichoderma species can withstand adverse environmental conditions and reproduce rapidly, hence they are highly aggressive phytopathogens [25]. Mycoparasitism is not the only mode of action but it is one of the biocontrol mechanisms deployed by Trichoderma. Trichoderma deploys different biocontrol mechanisms, which could be direct (enzymes, mycoparasitism, fungicides, bioactive and even volatile compounds) and indirect (competition, rhizosphere competence, and priming of plant immunity) [24,26,27].
The Discovery and use of novel phytochemicals have emerged as an important resource of new agrochemicals for the control of many types of plant pathogens. For example, the Cucurbitaceae family consists of herbaceous climbers of annual plants with over nine hundred species [28]. Citrullus colocynthis (L.) Schrad, of this family, is broadly distributed around the warmer areas of the world including West Asia and Tropical Africa and is widely distributed in India, Pakistan, and Saudi Arabia [29,30]. C. colocynthis fruits are commonly known as ground melons [29,31]. Phytochemicals from the fruits, roots, and leaves of C. colocynthis, were shown to have antibacterial activity substances from aqueous and hydromethanolic extracts of leaves, roots, and fruits against Klebsiella pneumonia, Bacillus stearothermophilus, and Staphylococcus aureus [32].
This work investigates the antimicrobial activity of the fungal biocontrol agent T. viride with extracts of fruits from C. colocynthis against the severe fungal pathogen FOL. The compatibility of this pair of treatments was also evaluated for efficacy and suitability of these two biocontrol agents for use on tomatoes under greenhouse conditions.

Collection of plant material and extraction
The fruits of C. colocynthis were harvested in and around Southern Western Ghats, Tirunelveli District, India during the summer seasons. The collected plants were authenticated and the voucher specimen (Collection number 966 and reference number BSI/CRC/Tech./ 2012-13) of this collection has been submitted to the herbarium of SPKCEES, Manonmaniam Sundaranar University, India. The extraction procedure followed by Visetson et al [33] with slight modifications [34]. The yields of fruit extracts (hexane, chloroform, methanol, and water) were 3.93. 2.76, 4.31, and 4. 28g respectively.

Fungal pathogen culture
FOL (NFCCI No: 745) was purchased from the National Fungal culture collection of India (NFCCI). Further, the culture was maintained in Potato Dextrose Agar (PDA) medium and incubated at the laboratory condition at 25 ± 3˚C. After 48 h, hyphal tips of fungi growing out from the collar region were cut and transferred onto PDA slants [35] and preserved at 24-26˚C in the Biological Oxygen Demand (BOD) incubator.

Growth inhibition assay
The Growth inhibition assay was performed by the method of Nene and Thapilyal, [36] with slight adjustments. Two milliliters of fruit extract prepared from each of the solvent extracts at different concentrations (1, 5, and 10%) was mixed with PDA medium. The PDA medium mixed with plant extract was poured into sterilized Petri plates at 15 mL/ plate and allowed to solidify. The plates were then inoculated with the 6 mm discs of a 5-day-old culture of FOL grown on PDA and incubated at room temperature (28 ± 3˚C). Petri plates were sealed with Parafilm™ and incubated at 27 ± 2˚C in a BOD incubator for 6 days. The percent inhibition of radial growth of FOL was calculated using Formula 1. Five replicates were used for each treatment and the experiment was repeated three times.
C = radial growth of FOL in the control plate, and T = radial growth of FOL with C. Colocynthis extract.

Analysis of fruit extract through FT-IR and GC-MS
The preliminary antifungal activity was performed with FOL with hexane, chloroform, methanol, and water extract of C. colocynthis. The greatest antifungal activity was observed in methanol and water extract and their chemical constituents were determined with FT-IR and GC-MS analysis.
Perkin Elmer Spectrum One equipped with an ATR-FTIR unit was used to measure the FTIR spectra of the extract in a wavelength range of 450-4000 cm -1 . The 16 scans were accumulated and averaged. The spectra were collected and analyzed using Spectrum software (Perkin Elmer).
Two hundred microliters of HPLC-grade methanol were dissolved with 200 μl of water extract and examined using a GC-MS spectrometer (Agilent Technologies, Santa Clara, CA). The compound structure, molecular weight, and molecular formula were identified by matching the sample spectrum to those in the database (NIST). The area % was generated using the total ion chromatogram (TIC) peak areas of the eluted compounds.

Maintenance and mass culture of T. viride
The T. viride (MTCC no: 800) was purchased from Microbial Type Culture Collection (MTCC). A pure culture of T. viride was maintained in a PDA medium and maintained at the laboratory condition at 25 ± 2˚C.
Sorghum seeds (100 g) were boiled up to 20 to 25 min to soften grains and were then spread out in a laminar airflow chamber to decrease the moisture content. Two grams of calcium carbonate were added (2 g/100 g of seeds) to remove the excess moisture. One-fourth volume of a 250 ml conical flask was filled with these grains (Fig 4) and autoclaved at 121˚C for 15 min. After cooling, the sorghum seeds were inoculated with a few discs of bio-agent (5 mm) from a 6-7 days old culture of T. viride conidia grown in PDA medium and incubated at 24 ± 2˚C in a BOD incubator for 7-12 days.

Compatibility of C. colocynthis fruit extract with T. viride
The compatibility was determined for T. viride and C. colocynthis fruit extracts (Methanol and water) by using a PDA medium. The PDA medium was mixed with the T. viride (2 mL/ 18 mL). Every single milliliter of fungal suspension contained 1 × 10 6 cfu/mL. The medium containing T. viride was poured into Petri plates and immediately after solidification, two sterile paper discs were placed at equidistance to each other on the seeded medium. Six μL of the 2% fruit extract (Methanol and water) after passing through bacterial filters was added to the first sterile filter paper disc and 6 μL of 0.01 mL of sterile distilled water was added to the second filter paper disc as control, respectively, and incubated at 28 ± 2˚C for 5 days. After 5 days, the inhibition zone was observed. The presence of an inhibition zone around the disc indicates incompatibility with fungal isolates and the absence of an inhibition zone indicates compatibility [37].

Efficacy of individual and combination of T. viride and fruit extract against pathogen
Compatibility result showed methanol extract was compatible with the biocontrol agent T. viride hence; the methanolic extract was used for further experiments. T. viride was assayed against FOL with the dual-culture method of Rajeev and Mukhopadhyay [38] with little modification. Discs (6 mm diameter) of the pathogen and the antagonist were cut from the edge of the 5-day-old culture and placed on the opposite side of Petri dishes containing PDA, 1 cm away from the edge. Three replications were maintained. Petri dishes were incubated for 4 days at 28 ± 2˚C and the mycelial growth of FOL was measured. Percent inhibition (PI) of mycelial growth was calculated using the formula suggested by Pandey et al. [39].
C. colocynthis fruit extract was tested individually and in combination with T. viride against FOL by dual culture technique [40]. Sterilized filter paper discs of 3 mm diameter were spotted with 6 μL of C. colocynthis fruit methanol extract. For combination, 3 μL of T. viride (2%) and C. colocynthis methanolic extract (2%) were spotted on sterilized filter paper discs (3 mm diameter) and placed 1 cm away from the edge of the plate containing PDA medium using pour plate technique with the pathogen (dosage of 1 × 10 6 cfu/mL). Four replicates were used for each treatment and the experiment was repeated three times. The plates were incubated at room temperature (28 ± 2˚C) for 7-days and the inhibition zone was measured and percent reduction over control was calculated.

Bio formulation
The talc-based formulation of T. viride was prepared with the adapted procedure of Srivastava et al. [41]. After 12 days, the conidia were harvested from the sprouted sorghum seeds and grounded in a laboratory blender to form a suspension. The inoculums were mixed with talcum powder to obtain a conidia concentration of 1 × 10 6 conidia/ml. Ten grams of carboxy methyl cellulose (CMC) was added to one kg of talc and mixed well. The formulations thus prepared were allowed to dry aseptically overnight (approximately 35% moisture content), then it was packed in sterile polythene bags and stored at 4˚C.
In the case of fruit extract, 20 mL of fruit extract was added to 100 g of sorghum seeds. Then, T. viride was inoculated and incubated for 12 days at 28 ± 2˚C and the formulation was prepared as previously described.

Effect of bio formulation mixtures on the incidence of wilt disease.
Potting medium (red soil: cow dung: vermiculate at 2:1:1, w/w/w) was autoclaved at 105˚C for 1 h and filled in pots. Then, the tomato (var.PKM1) seedlings (2-3 true leaf stage) produced in vermiculite were transplanted into autoclaved pot mixture in the surface sterilized (1% mercuric chloride) pots (15 cm diameter, 750 ml volume) at 1 seedling/pot. In all treatments, the talc-based bio formulation mixture was applied as 1:20 (w/w) and foliar spray (20 g/l). Talc-based bio formulations were applied basally at the rate of 10 g/pot. The talc-based bio formulation mixtures were applied 45 days after planting and after 1 day, the plants were inoculated with the spore suspension of FOL(1 × 10 6 conidia/ml). The pathogen alone inoculated served as a control. Soil drenching of 0.1% carbendazim was included as a chemical check (Positive control). Control plants received only the talcum powder with adhesive. Ten days after inoculation, observations on the development of wilt symptoms were made. Three replications (three pots per replication) were maintained and the pots were arranged in a randomized manner.
The measurement of disease index (DI) was determined using the given formula (2) [42].
Where A = Sum of the individual scores; B = Total leaves observed; C = Maximum score. The disease reduction was calculated using the given formula (3).
The disease severity was recorded on a 0-4 scale as described by Song et al. [43] where zero represents no infection and four denotes complete infection. The 0-4 scale of the disease severity was classified as follows: 0-No infection. 1-Slight infection, which is about 25% of full scale, one or two leaves turned yellow. 2-Moderate infection, two or three leaves turned yellow, 50% of leaves wilted. 3-Extensive infection, all the leaves turned yellow, 75% of leaves wilted and growth was inhibited. 4-Complete infection, the leaves of the whole plant turned yellow, 100% of leaves wilted, and the plants died.
The percentage of disease incidence was determined using the formula (4) given by Song et al. [43].
2.9.2 Assay of induced defense enzymes. T. viride and C. colocynthis fruit extract were used single and in combinations for the induction of defense reactions in tomatoes. The bio formulations treated seeds were sown in earthen pots (size: 0.35 m diameter, 0.50 m height; volume of soil: 0.04 m3) filled with sterilized potting soil at five seeds per pot. Forty-five days after sowing, foliar spray with bio-formulation mixtures was done as described earlier. Bio-formulation-treated plants were divided into two sets. In the first set, treated plants were challenge inoculated with FOL (at 1 day after foliar spray) and in the second set, treated plants were not challenged with the pathogen. Plants without prior treatment of bio formulations were inoculated with the pathogen. The plants neither treated with bio formulation nor challenged by the pathogen were kept in control. Three replications were maintained in each treatment; each replicate consisted of five pots and in each pot, one plant was maintained. The experiments were conducted using completely a randomized block design on a greenhouse bench. The humidity in the greenhouse was maintained at around RH 70%. The temperature was adjusted to 26 ± 2˚C (day)/20 ± 2˚C (night).
Leaf tissues were collected after 5 days of pathogen inoculation. Three plants were sampled from each replication of the treatment separately and were maintained for biochemical analysis. Leaf samples were homogenized with liquid nitrogen in a pre-chilled mortar and pestle. One gram of leaf sample was homogenized with 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) at 4˚C. The homogenate was centrifuged for 20 min at 10,000 rpm. The supernatant was used as a crude enzyme extract for assaying PO [44] and polyphenol oxidase (PPO) [45]. An enzyme extracted in 0.1 M sodium citrate buffer (pH 5.0) was used for the estimation of chitinase [46] and β-1,3-glucanase.

Statistical analysis
The data on the effect of the treatments on the growth of pathogens, severity of diseases, and activity of enzymes in rice plants were analyzed with analysis of variance (ANOVA), and treatment means were compared by Tukey's-family error test (P < 0.05) for pairwise comparison using Minitab 1 16 software package. The data on in vitro bacterial growth inhibition, disease incidence, disease index, and suppression efficiency were arcsine transformed before undergoing statistical analysis.

Growth inhibition assay
Fruit extracts of C. colocynthis using hexane, chloroform, methanol, and water at various concentrations (2.5, 5, and 10%) were evaluated for antimicrobial activity against the pathogen FOL. All the extracts at 10% concentration were effective in inhibiting the radial growth of FOL, compared to the control. The methanolic fruit extract of C. colocynthis at 10% was most effective in inhibiting the mycelial growth of FOL (12.32 mm) lowed by 10% of water, chloroform, and hexane with 23.61, 35.62, and 40.48 mm respectively. In contrast, FOL at control had mycelial growth of 90.0 mm ( Table 1). All the treated concentration of C. colocynthis was significantly different from the control (F 11,28 = 39.26, P � 0.001).

FT-IR and GC-MS analysis
The FT-IR spectrum of methanolic extract (Fig 1A) shows the presence of several bands corresponding to the wave numbers 3343, 2920, 1630, 1372, 1043, and 877 cm -1 . The band detected at 3343 cm -1 arises due to the O-H stretch of alcohols and phenols. The band observed at 2920 cm -1 formed due to the O-H stretch of carboxylic acids and also due to the C-H stretch of alkanes. The N-H bend of primary amines was found at 1630 cm -1 . The bands arise at 1372 cm -1 attributed to the C-H bend of alkenes and the stretch of N-O due to the nitro groups. The carboxylic acids, alcohols, esters, and ethers have O-O stretch at 1043 cm -1 . The out-of-plane (oop) stretch of aromatics (C-H) was found at 877 cm -1 and also the N-H wag of primary and secondary amines was found in this extract. Similarly, the FT-IR spectrum of water extract (Fig 1B)  The compounds present in methanolic and water extract of C. colocynthis were identified and the mass spectrum of the analysis was confirmed with comparisons to the NIST library. The retention time (RT), compound name, molecular weight (g/mol), molecular formula, peak area percentages, and chemical structures were presented in Figs 2 and 3.

Compatibility of C. colocynthis fruit extract with T. viride
The antagonist fungi T. viride and C. colocynthis methanolic extract were tested for their compatibility in vitro. The absence of an inhibition zone around the paper disk was observed in C. colocynthis methanolic extract. This result indicated that the biocontrol agent T. viride was compatible with C. colocynthis methanolic extract. On the other hand, C. colocynthis water extract was not compatible with the biocontrol agent, showing an inhibitory effect on T. viride.

In vitro screening of fungal antagonists and C. colocynthis against FOL
Treatments with T. viride and C. colocynthis methanolic extract were tested individually and in combination to assess the radial growth of FOL. Compared to the control plates (Fig 6A and  6B) all the treatments were effective in reducing the mycelial growth of the pathogen FOL. The dual culture results show that T. viride alone reduced the mycelial growth of FOL by 26 mm (Fig 6C). After 9 days of In-vitro screening of T. viride were observed against the wilt pathogen FOL (Fig 6D). However, the combined application of T. viride and C. colocynthis methanolic extract resulted in the highest inhibition of 2.5 mm with least mycelial growth of 14.21 mm and accounting for 82.92% reduction of FOL mycelial growth compared to control. The control plates recorded the highest mycelial growth of 83.24 mm ( Table 2). All the treatment concentrations were significantly different from the control (F 4,10 = 34.59, P � 0.001).

Effect of bio formulation mixtures on the incidence of wilt disease
Talc-based formulations of T. viride and C. colocynthis methanolic extract were tested individually or in combination, for their efficacy against FOL under pot culture conditions. The biocontrol agents and C. colocynthis methanolic extract significantly reduced the wilt disease by 47.23 and 41.05% compared to the control (Table 3). Noticeably, a combination of T. viride and C. colocynthis methanolic extract significantly lower the disease incidence (Fig 7) by 21.92%. Whereas, carbendazim at 0.1% decreases the disease incidence by 32.53% only. All the treatment concentrations were significantly different from to control (F 4,10 = 30.29, P � 0.001). However, C. colocynthis methanolic extract was not significant to T. viride (F 4,10 = 30.29, P � 0.201) and carbendazim (F 4,10 = 30.29, P � 0.052).
The disease index in control was 81.36% and decreased to 52.71, 45.43 and 27.02% with the individual treatment of T. viride and C. colocynthis methanolic extract and in combination respectively. The result revealed that all the treatments were significant difference compared to the control (F 4,10 = 36.24, P � 0.001). But, C. colocynthis methanolic extract was not significant to T. viride (F 4,10 = 36.24, P � 0.108). Interestingly, a combination of antagonistic fungi and C. colocynthis methanolic extract resulted in a prominent upsurge in the percentage of disease reduction over control (66.78%) than the chemical carbendazim (49.32%).

Assay of induced defense enzymes
Defense-related enzymes viz., peroxidase, polyphenol oxidase, β-1,3-glucanase, and chitinase showed higher activity in all treatments compared with non-inoculated control.
Peroxidase enzyme activity was strongly induced in the combined treatment of T. viride and C. colocynthis methanolic extract followed by the treatment of C. colocynthis extract, T. viride, carbendazim and inoculated control (Fig 8A). Peroxidase enzyme activity in non-inoculated control (F 5,12 = 21.31, P � 0.001) was significantly different than all the treatments. However, the treatment of carbendazim did not yield significant results compared to the treatment of T. viride (F 5,12 = 21.31, P � 0.063) and inoculated control (F 5,12 = 21.31, P � 0.058).
Similarly, the polyphenol oxidase enzyme was also increased in the combined treatment of T. viride and C. colocynthis methanolic extract (Fig 8B). Polyphenol oxidase enzymes induced in non-treated control plants are considered to be statistically significant when compared to other treatments (F 5,12 = 27.87, P � 0.001). Pathogen-inoculated plants were gradually increased in PPO with 2.08, 1.53, 1.42, and 1.2 absorbance/min/g by the treatment of a

PLOS ONE
Induced systematic resistance in Tomato combination of T. viride and C. colocynthis methanolic extract, C. colocynthis extract, carbendazim, and T. viride.
As found in PO and PPO, an increase in β-1,3-glucanase enzyme activity was observed in all the treatment concentrations. β-1,3-glucanase enzyme activity in pathogen non-inoculated control was 21 μg min -1 g -1 and increased to 28, 32, 37. 38 and 46 μg min -1 g -1 with the treatment of pathogen inoculated control, T. viride, carbendazim, C. colocynthis, and a combination of T. viride and C. colocynthis methanolic extract (Fig 8C). The results revealed that all the treatments increased the β-1,3-glucanase enzyme activity and it illustrates a significant difference compared to the control (F 5,12 = 26.33, P � 0.001). Furthermore, the treatment of C. colocynthis and carbendazim (F 5,12 = 26.33, P � 0.952) was not significant to each other.
Results obtained from chitinase activity observed that all the treatment concentrations triggered the chitinase activity. The result demonstrates that combined treatment of T. viride and C. colocynthis methanolic extract shows an increased in chitinase activity (Fig 8D) at 2.28 μg min -1 g -1 . Plants exposed with the treatment of inoculated control, T. viride, carbendazim, and C. colocynthis, whose chitinase activity was increased to 0.8, 1.25, 1.32, and 1.43 μg min -1 g -1 respectively. Chitinase activity in all the treatments shows significantly different judge against its non-inoculated control (F 5,12 = 24.05, P � 0.001). But, the chitinase activity of carbendazim was not significant in the treatment of C. colocynthis methanolic extract (F 5,12 = 24.05, P � 0.362) and T. viride (F 5,12 = 24.05, P � 0.771).

Discussion
Biological control agents developed for agricultural crop pathogens, especially soil-borne plant pathogens from botanicals, in combination with micro-organisms are gaining momentum as a viable system in controlling plant pathogen diseases with more native and environmentally acceptable alternatives to the current heavy dependence upon harmless chemical treatments.  Controlling crop diseases by application of plant products was previously suggested by many researchers [47]. In the current study, we assessed the antifungal activity of C. colocynthis fruit extracts (Hexane, chloroform, methanol, and water) against a vascular wilt pathogen. Methanol and water extracts were effective in reducing the mycelial growth of FOL. Methanol extract of C. colocynthis controlled the FOL by 44%. Many researchers have described the antifungal activity of different botanicals against phytopathogenic fungi FOL [48,49].
Crop Survey report of 2012, reviewed F. oxysporum ranking as the third most destructive plant pathogen [50]. It causes severe yield losses of both field and greenhouse tomato crops [11]. Treatments to control Fusarium wilt in tomato crops have become one of the most challenging tasks in terms of management [51].
On the other hand, plant exposure to beneficial microbes also induced systemic resistance [52]. The mycoparasitic soil fungi, T. viride, is the most extensively used biocontrol agent and it plays an important role in mycoparasitism, antibiosis, and production of elicitors in plants [53]. In this study use of the antagonistic fungi, T. viride inhibits the pathogen FOL by 68%. Similar results by John et al. [54] and Singh and Kumar [48], reported that T. viride effectively controlled the fungal pathogen FOL. When combined the fruit extract from C. colocynthis and T. viride resulted in effective FOL control. The combination of the two was also compatible. Muthu Kumar et al. [55] reported that T. viride was also compatible with the extract of Allium sativum × Allium cepa.
An essential principle for a successful application of T. viride is the preparation of microbial biomass with high effectiveness and an inexpensive ingredient. The advances in biological control for crops is being contingent not only on the antagonist effectiveness of biocontrol agents but also on the costs of mass production. In this report, low-cost sorghum seeds were shown to be a suitable and affordable substrate to support T. viride growth for mass production. Thangavelu et al. [56], described five different organic substrates (rice bran, rice chaffy grain, farmyard manure, banana pseudo stem, and dried banana leaf) that could also be used as a substrate for the growth of Trichoderma sp, demonstrating that this technology is within reach of farmers in most countries. In parallel, Srivastava et al, (2010) [41] used six different substrates (Jhangora, mandua, sorghum, polygonum, rice straw, wheat straw) for mass cultivation of Trichoderma sp. showing higher biomass production in sorghum seeds next to Jhangora and mandua. Our results show that T. viride requires a minimum period of 7-12 days to grow on the substrate (Sorghum seeds). Thus, strongly suggests that it is possible to use sorghum seeds as an inexpensive substrate to produce and harvest T. viride for use in agriculture fields to suppress phytopathogens.
Our results show that tomato plants treated with T. viride display 29% protection against FOL. Soil application of the biocontrol agent T. viride successfully reduces soil-borne pathogens [57,58]. Dubey et al. [59] reported that Trichoderma sp viz. T. viride, T. harzianum, and T. virens inhibit the mycelial growth of FOL. Significantly improved disease protection of 67% against FOL was observed only in plants treated with both C. colocynthis extract and T. viride. Similar reports have been published that support successful biocontrol agents used in combination which were significantly better than a single agent [41,55,58,60].
Priming for enhanced defense is a common feature of induced resistance and can explain the broad-spectrum efficiency that is typical for many induced resistance phenomena. In this current study, tomato seedlings treated with C. colocynthis extract and T. viride were evident from the increased activity of defense-related enzymes. The defense-related enzymes viz., PO, PPO, chitinase, and β-1,3 glucanase stimulate induced systemic resistance to protect themselves against pathogen attack [4,61]. The induction of defense-related enzymes is a sign of induced systemic resistance in plants. Defense-related enzymes like PO and PPO have been shown to have direct antifungal activities [62]. Plants PO are generally secreted into cell walls and vacuoles or the surrounding medium [63,64]. Moreover, PO was described to play a part in the production of reactive oxygen species, which are active in the signal transduction pathway of plant defense mechanism [65].
Production of PO (1.92 absorbance/min/g), PPO (2.08 absorbance/min/g), β-1,3 glucanase (46 μg/min/g), and chitinase (2.28 46 μg/min/g) was significantly higher in tomato plant treated with C. colocynthis extract and T. viride, as compared to un-inoculated control. Chen et al. [66] observed that PO and PPO activity was enhanced in the biocontrol of cucumber Fusarium wilt by the treatment of Bacillus subtilis B579. Hence, our study supports the hypothesis that the reductions in disease index in tomato plants treated with fungal pathogen FOL are due to the induced systemic resistance which suppresses the FOL by the production of defense-related enzymes.

Conclusion
As an endnote, our study showed that a combination of methanolic fruit extract of C. colocynthis and T. viride reduced Fusarium wilt disease significantly better than either a single application of C. colocynthis or T. viride, alone. This was due most likely to induced defense enzyme activity. Induction of resistance described in the current study is an easy and less expensive technique for procuring maximum protection in fungal pathogen disease management.
Supporting information S1 File. All the supporting information including Fruit of C. colocynthis, Mass culture of T. viride using sorgum seeds and growth rate of plant (A) Control plant (B) T. viride inoculated plant was displayed (S1 Fig 1-3). Also, the growth rate comparison of T. viride and F. oxysporum. Means (±(SE) standard error) indicate no significant difference (P�0.05) according to a Tukey test was displayed (S1 Table). (DOCX) S1 Graphical abstract. (DOCX) S1 Raw data. (XLSX)